1. Field of the Invention
The invention is directed to a method and arrangement for realizing a PALM microscope with optimized photoactivation for realizing a higher image rate.
2. Description of Related Art
Selective plane illumination microscopy (SPIM) has been described as a microscopy method, for example, in Stelzer et al. [1-4]:
[1] Stelzer et al., Optics Letters 31, 1477 (2006).
[2] Stelzer et al., Science 305, 1007 (2004).
[3] DE 102 57 423 A1
[4] WO 2004/0530558 A1
Like confocal laser scanning microscopy, SPIM, as a widefield technique, allows three-dimensional objects to be recorded in the form of optical sections, the advantages residing primarily in speed, reduced bleaching out of the sample, and expanded depth of penetration. For this purpose, generally, fluorophores in the sample are excited by laser light which is shaped as a light sheet. The light sheet can be scanned through the sample. A spherical PSF can be generated through the (computational) combination of images recorded from different angles. As a rule, its extent is determined by the lateral resolution of the detection lens that is used, which generally limits the optical resolution that can be achieved in the conventional SPIM method.
Breuninger et al., Optics Letters, Vol. 32, No. 13, 2007, describe the SPIM method with a periodically structured light sheet. The fluorescence excitation is carried out at the locations of high intensity by means of the periodically structured light sheet. The structuring is used for suppressing scattered light from out-of-focus planes and for increasing resolution by structured illumination (see below: Heintzmann et al.).
The method of photoactivated light microscopy (PALM) is described in WO2006/127692. The method is based on the photoactivation of individual molecules which are separated from one another depending on the dimensions of the detection PSFs and highly precise localization thereof by fluorescence detection.
The PALM method, as it is described in WO2006/127692, uses substantially the following main steps to generate a microscope image with a higher optical resolution compared to the standard microscope:
1. Photoactivation of individual molecules: The fluorescence characteristics of the molecules are changed through activation (on/off switching, change in the emission spectrum, . . . ). The activation is carried out in such a way that the distance between activated molecules is greater than or equal to the optical resolution of the standard microscope (given by the Abbe limit).
2. Excitation of the activated molecules and localization of the molecules by a spatially resolving detector.
3. Deactivation of the activated molecules.
4. Repeating steps 1-3 and superimposing the localization points from step 2 which were acquired from different iterative steps to form a high-resolution image.
The activation is preferably carried out in widefield illumination and is statistically distributed. Through the choice of activation energy, it is attempted to achieve (1) as few molecules as possible/no molecules (2) with overlapping Airy disks on the camera (see FIG. 1a). However, overlapping Airy disks are still present and cannot be evaluated ((3) in FIG. 1b). Accordingly, there are regions in which the distance between the activated molecules is larger or very much larger than the Airy disks on the camera (4). Because of the statistical activation of the molecules, approximately 10,000 individual images must be evaluated for generating a high-resolution image to determine the positions of the molecules. Large amounts of data must be processed for this purpose, and measurement is slowed down (approximately 1 minute per high-resolution image). Computation of the individual images to form a high-resolution image requires about 4 hours.
There are difficulties involved in applying the PALM method in three-dimensional imaging because molecules outside of the focus plane are also activated and are therefore bleached and their fluorescent light cannot be used for imaging. Above all, autofluorescent light which is considered an interference signal and causes an extreme reduction in contrast is excited in the entire focus cone in biological samples. This hinders the recording of a z-scan so that three-dimensional imaging of the sample cannot be achieved.
WO2006/127692 describes the use of multiphoton excitation to prevent photoactivation and interfering autofluorescence outside the focus plane. However, the technology is complicated in this arrangement. For example, the dyes (PA-GFP) must be nonlinearly activatable and high intensities must be used which can result in damage to the dye or sample.
Another method that is used to prevent autofluorescence problems is to combine the PALM method with the TIRF technique in which the excitation volume in z-direction is kept very small due to limiting to evanescent waves. However, three-dimensional imaging is not possible with TIRF.
In principle, PALM initially offers only an improved lateral resolution because of the spatially resolved detection. The axial resolution is primarily determined by the extent of the detection PSF that is used. This is another reason for combining PALM with TIRF, which offers a high axial resolution (see also WO2006/127692).
Aside from PALM, other resolution-enhancing methods are known in which the sample is illuminated in such a way that a region detectable through fluorescence is formed which is smaller than corresponds to the Abbe diffraction limit. This is accomplished through a nonlinear interaction based on different methods:
De-excitation of previously excited molecules by stimulated emission (STED, Klar and Hell, Opt. Lett. 24 (1999) 954-956)
De-excitation of previously excited molecules through further excitation into a higher non-fluoresceable state (Excited State Absorption, Watanabe et al., Optics Express 11 (2003) 3271)
Depletion of the ground state by populating with triplets (Ground State Depletion, Hell and Kroug, Appl. Phys. B 60 (1995), 495-497)
Switching a dye between a fluorescing and non-fluorescing state, a less fluorescing state or a fluorescing state characterized in some other way (such as with a different emission wavelength, polarization) (Hell, Jakobs and Kastrup, Appl. Phys. A 77 (2003) 859-860).
In general, these are point-scanning methods having disadvantages with respect to fast data acquisition. Further, the sample is unnecessarily stressed in out-of-focus regions.
As another concept for increasing resolution, Heintzmann et al. (R. Heintzmann, T. M. Jovin and C. Cremer, “Saturated patterned excitation microscopy—a concept for optical resolution improvement”, JOSA A 19, 1599-1609 (2002)) suggest a nonlinear process in the form of direct saturation of a fluorescence transition. The increased resolution is based on structured illumination of the sample with periodic grid shapes so that there is a transfer of high object space frequencies into the optical transfer function domain of the microscope. The transfer can be reconstructed indirectly through theoretical post-processing of the data. It is also considered disadvantageous in these methods that the sample is unnecessarily stressed in out-of-focus regions because the structured illumination is performed throughout the entire sample space. Further, the method cannot currently be used with thick samples because the fluorescence excited outside of the focus reaches the detector as a background signal and accordingly reduces the dynamic range.